title: A new real time PCR-based assay for diagnosing Renibacterium salmoninarum in rainbow trout (Oncorhynchus mykiss) and comparison with other techniques.
abstract: Bacterial Kidney Disease of salmonid is caused by a slow-growing gram-positive bacterium, Renibacterium salmoninarum. This bacterium lives both extra-cellular and intra-cellular in the host. Serological and molecular diagnostic methods to detect the bacterium major surface protein antigen p57 have been developed. In the present work, a newly developed quantitative Reverse Transcriptase-PCR (RT-QPCR), using self-quenched fluorescent primer (Lux), a nested PCR (NPCR), a commercial ELISA and recently commercially available Immune-chromatographic strip test(IC-Strip) were compared for their ability to detect BKD in kidney tissue samples obtained from experimentally infected fish. ELISA test resulted to be rapid, simple and indicative for the bacterial load. The IC-Strip test had similar characteristics for bacterial detection. Both tests are a good option for rapid and relatively inexpensive screening studies, despite the one and two log decrease in bacterial detection limits compared to NPCR and RT-QPCR, respectively. The use of Lux primers in the newly developed RT-QPCR revealed to be a cost-effective alternative to other fluorescence-based PCR techniques. The option of generating a melting temperature curve with the real time PCR instrument confirmed the specificity of the PCR product. The RT-QPCR technique had the advantage of detecting low numbers of viable bacterial mRNA which implied a higher capacity of detecting chronically infected animals. For instance, some fish in the group infected by cohabitation had very low bacterial load and were only detected by this technique.
authors.affiliation: Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Zaragoza 50013, Spain. Nabilhk@unizar.es
authors.forename: Nabil
authors.initials: N
authors.lastname: Halaihel
authors.forename: Daniel
authors.initials: D
authors.lastname: Vendrell
authors.forename: Imanol
authors.initials: I
authors.lastname: Ruiz-Zarzuela
authors.forename: Ignacio
authors.initials: I
authors.lastname: de Blas
authors.forename: José Luis
authors.initials: JL
authors.lastname: Alonso
authors.forename: Olivia
authors.initials: O
authors.lastname: Gironés
authors.forename: Tania
authors.initials: T
authors.lastname: Pérez
authors.forename: José Luis
authors.initials: JL
authors.lastname: Muzquiz
mesh_terms: D000193:Actinomycetales Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005393:Fish Diseases / Q000175:diagnosis* / Q000382:microbiology; D008835:Micrococcaceae / Q000235:genetics / Q000302:isolation & purification*; D017686:Oncorhynchus mykiss / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011897:Random Allocation; D012680:Sensitivity and Specificity